The actinobacterial acI lineage is among the most successful and ubiquitous freshwater bacterioplankton found on all continents, often representing more than half of all microbial cells in the lacustrine environment and constituting multiple ecotypes. However, stably growing pure cultures of the acI lineage have not been established despite various cultivation efforts based on ecological and genomic studies on the lineage, which is in contrast to the ocean from which abundant microorganisms such as Prochlorococcus, Pelagibacter, and Nitrosopumilus have been isolated. Here, we report the first two pure cultures of the acI lineage successfully maintained by supplementing the growth media with catalase. Catalase was critical for stabilizing the growth of acI strains irrespective of the genomic presence of the catalase-peroxidase (katG) gene. The two strains, representing two novel species, displayed differential phenotypes and distinct preferences for reduced sulfurs and carbohydrates, some of which were difficult to predict based on genomic information. Our results suggest that culture of previously uncultured freshwater bacteria can be facilitated by a simple catalase-supplement method and indicate that genome-based metabolic prediction can be complemented by physiological analyses.

In order to investigate the importance of spatial and environmental factors on the structure and diversity of bacterial communities, high-resolution 16S rRNA gene tag pyrosequencing was applied to bacterial communities in the littoral sea. Seawater samples were prepared from seven different stations in the South Sea of Korea, the marginal sea in the western Pacific Ocean, and were divided into three groups according to distances from the coastline. The majority of 19,860 sequences were affiliated with Alphaproteobacteria (58.2%), Gammaproteobacteria (7.9%), and Bacteroidetes (13.9%). The bacterioplankton community at each station was highly diverse and varied among the samples. Major bacterial lineages showed different niche preferences among three locational groups. Alphaproteobacteria was the most abundant bacterial class, and it harbored the most frequently recorded operational taxonomic units (OTUs) in all sampling stations. However, dominant groups at the order levels showed a clear difference among the samples. The SAR11 clade was more abundant in coastal waters while the Roseobacter clade prevailed at stations far away from the coastline. Furthermore, members of Actinobacteria and Cyanobacteria also exhibited spatial variability. The OM1 clade in Actinobacteria constituted a predominant fraction in coastal samples, but it was essentially absent at the distal stations closer to open ocean. In contrast, Synechococcus was the predominant taxon in the distal samples, accounting for 7.1–19.5%, but was hardly detected in coastal waters, representing less than 0.7%. In Bacteroidetes, NS5 and NS9 groups tended to inhabit coastal waters while the genera Polaribacter and Ulvibacter were more abundant in distal stations. Clustering analysis and principle coordinates analysis based on OTU data indicated that bacterial communities in the studied area were separated into three groups that coincided with locational grouping. Statistical analysis showed that phosphate and dissolved oxygen concentration had a significant influence on the bacterial community composition.

24Unlike the ocean from which abundant microorganisms with streamlined genomes such as 25 Prochlorococcus, Pelagibacter, and Nitrosopumilus have been isolated, no stable axenic bacterial cultures are 26 available for the ubiquitous freshwater actinobacterial acI lineage. The acI lineage is among the most successful 27 limnic bacterioplankton found on all continents, often representing more than half of all microbial cells in the 28 lacustrine environment and constituting multiple ecotypes. Dilution-to-extinction culturing followed by whole-29 genome amplification recently yielded 20 complete acI genomes from lakes in Asia and Europe. However, stably 30 growing pure cultures have not been established despite various efforts at cultivation using growth factors 31 predicted from genome information. Here, we report two pure cultures of the acI lineage successfully maintained 32 by supplementing the growth media with catalase. Catalase was critical for stabilizing growth by degrading 33 hydrogen peroxide, irrespective of the genomic presence of the catalase-peroxidase (katG) gene, making the acI 34 strains the first example of the Black Queen hypothesis reported for freshwater bacteria. The two strains, 35 representing two novel species, displayed differential phenotypes and distinct preferences for reduced sulfurs 36 and carbohydrates, some of which were difficult to predict based on genomic information. Our results suggest 37 that culture of previously uncultured freshwater bacteria can be facilitated by a simple catalase-supplement 38 method and indicate that genome-based metabolic prediction can be complemented by physiological analyses. 39 40 3The acI lineage of the phylum Actinobacteria is the most abundant and cosmopolitan bacterial group in 41 most freshwater environments. Since the acI lineage was first suggested to denote an abundant monophyletic 42 actinobacterial group exclusively found in freshwater environments (1-3), many studies have demonstrated the 43 ubiquity and prevalence of the acI lineage in diverse freshwater ecosystems on all continents (4-10). 44 Studies employing fluorescence in situ hybridization (FISH) and PCR-based 16S rRNA gene sequence 45 profiling showed that the acI lineage and its subgroups exhibit specific distributions depending on the season, 46 depth, and habitat characteristics and that there are >10 monophyletic tribes belonging to three sublineages (acI-47

Reduced glutathione (GSH) is an essential metabolite that performs multiple indispensable roles during the development of Dictyostelium. We show here that disruption of the gene (gcsA-) encoding y-glutamylcysteine synthetase, an essential enzyme in GSH biosynthesis, inhibited aggregation, and that this developmental defect was rescued by exogenous GSH, but not by other thiols or antioxidants. In GSH-depleted gcsA- cells, the expression ofa growth-stage-specific gene (cprD) was not inhibited, and we did not detect the expression of genes that encode proteins required for early development (cAMP receptor, carA/cAR1; adenylyl cyclase, acaA/ACA; and the catalytic subunit of protein kinase A, pkaC/PKA-C). The defects in gcsA cells were not restored by cAMP stimulation or by cAR1 expression. Further, the expression of yakA, which initiates development and induces the expression of PKA-C, ACA, and cAR1, was regulated by the intracellular concentration of GSH. Constitutive expression of YakA in gcsA- cells (YakA(OE)/gcsA-) rescued the defects in developmental initiation and the expression of early developmental genes in the absence of GSH. Taken together, these findings suggest that GSH plays an essential role in the transition from growth to development by modulating the expression of the genes encoding YakA as well as components thatact downstream in the YakA signaling pathway.

a b s t r a c tWe investigated the function of homeodomain-containing protein Hbx4 in Dictyostelium discoideum. Hbx4-overexpressing cells (Hbx4 OE ) displayed defects in growth rate and cytokinesis and showed differences in slug motility and cell-type proportioning from KAx3. Furthermore, the overexpression of Hbx4 inhibited the induction of cadA, which encoded the Ca 2+ -dependent cell adhesion molecule DdCAD-1, despite expression of csaA and gpaB. The electrophoretic mobility shift assay showed that the promoter of cadA contained the Hbx4-binding site. Moreover, constitutively expressed DdCAD-1 in Hbx4OE rescued the defects in cytokinesis and development. These results suggest that Hbx4 modulates DdCAD-1-mediated cytokinesis and cell-type proportioning.